Novel human membrane protein

ABSTRACT

The present invention provides a novel human integral membrane (IMP) and polynucleotides which identify and encode IMP. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding IMP and a method for producing IMP. The invention also provides for agonists, antibodies, or antagonists specifically binding IMP, and their use, in the prevention and treatment of diseases associated with expression of IMP. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding IMP for the treatment of diseases associated with the expression of IMP. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding IMP.

FIELD OF THE INVENTION

[0001] This invention relates to nucleic acid and amino acid sequencesof a novel integral membrane protein, IMP, and to the use of thesesequences in the diagnosis, prevention, and treatment of disease.

BACKGROUND OF THE INVENTION

[0002] Membrane proteins are divided into two groups based upon the easewith which the proteins can be removed from the membrane. Extrinsic orperipheral membrane proteins can be removed using extremes of ionicstrength or pH, the use of urea or other disruptors of proteininteractions. Intrinsic or integral membrane proteins are released onlywhen the lipid bilayer of the membrane is dissolved by detergent.Extrinsic membrane proteins comprise the constituents of thecytoskeleton such as spectrin and actin. Many cytoskeletal proteins arebound directly to integral membrane proteins or are bound indirectly viaother proteins such as ankyrin. Cytoskeletal proteins control the shapeand dynamics of the cell membrane through their interactions with motorproteins such as myosin and dynein.

[0003] The majority of known integral membrane proteins aretransmembrane proteins which have an extracellular, a transmembrane, andan intracellular domain. Transmembrane proteins are typically embeddedinto the cell membrane by one or more regions comprising 15 to 25hydrophobic amino acids which are predicted to adopt an α-helicalconformation. Transmembrane proteins are classified as bitopic (or TypesI and II) and polytopic (or Types III and IV) [Singer, S. J. (1990)Annu. Rev. Cell Biol. 6:247-96]. Bitopic proteins span the membrane oncewhile polytopic proteins contain multiple membrane-spanning segments. Asmall number of integral membrane proteins, termed monotopic proteins,are partially embedded in the membrane (i.e., they do not span the lipidbilayer). Monotopic proteins may be inserted into the bilayer by ahydrophobic hairpin loop or may be attached to the membrane via boundlipid.

[0004] A well characterized monotopic protein is the erythrocyte band7.2b protein also termed stomatin [Salzer, U. et al. (1993) Biochem.Biophys. Acta 1151:149-52]. Stomatin is absent in the erythrocytes ofpatients with hereditary stomatocytosis, an autosomal dominant hemolyticanemia. Hereditary stomatocytosis is characterized by abnormal membranepermeability to univalent cations (e.g., Na⁺ and K⁺) which leads toswelling and lysis of erythrocytes [Stewart, G. W. et al. (1993)Biochim. Biophys. Acta 1225:15-25]. It has been proposed that stomatinregulates an ion channel by acting as a plug which blocks a channelwhich is usually latent or tight in normal cells. Proteins whichcross-react with anti-stomatin antibodies and mRNA whichcross-hybridizes with stomatin gene sequences are found in a widevariety of human cell types and tissues suggesting that stomatin is awidely distributed regulator of transmembrane cation fluxes (i.e.,stomatin's functions are not limited to erythroid functions) [Stewart,G. W. et al. (1992) Blood 79:1593-1601 and Hiebl-Dirschmied, C. M. etal. (1991) Biochim. Biophys. Acta 1065:192-202]. In addition to its rolein ion transport, it has been proposed that stomatin acts to maintain anasymmetric distribution of phospholipids in erythrocytes and other celltypes [Desneves, J (1996) Biochem. Biophys. Res. Comm. 224:108-14]. Theexpression of stomatin has been shown to be up-regulated bydexamethasone and interleukin 6 in a human amniotic cell line suggestinga role for stomatin in protection of cells from oxidative stress[Snyers, L. and Content, J. (1994) Eur. J. Biochem. 223:411-18].

[0005] Stomatin has been shown to bind to the erythrocyte cytoskeletonvia its carboxyl region [Stewart G. W. et al. (1992), supra], and thusstomatin may play a role in the regulation of ion channels by providinga link between cyto skeletal proteins and ion channels. Interestingly,stomatin shares structural similarity to the central portion of theCaenorhabditis elegans (C. elegans) MEC-2 protein, a protein necessaryfor mechanosensation [Huang, M. et al. (1995) Nature 378:292-5]. MEC-2has been shown to link the mechanosensory channel and microtubulecytoskeleton of the touch receptor neurons. It has been proposed thatthis linkage permits the opening of the mechanosensory channel viamicrotubule displacement. Stomatin has been shown to share homology witha domain in another C. elegans protein, the UNC-24 protein. UNC-24 isrequired for normal locomotion in C. elegans [Barnes, T. M. et al.(1996) J. Neurochem. 67:46-57] and it has been proposed that UNC-24modulates directly or indirectly an ion channel.

[0006] The regulation of ion transport is an extremely importantphysiologic function as ion transport is involved in a wide variety ofcell functions including electrical excitability (i.e., action potentialpropagation), signaling, sensory transduction, control of Ca²⁺permeability via voltage-dependent Ca²⁺ channels, and in the control ofthe volume of intracellular and extracellular compartments. Inheriteddisorders of ion transport include hereditary stomatocytosis, cysticfibrosis, and a variety of hemolytic anemias (e.g., hydrocytosis andxerocytosis).

[0007] The discovery of molecules related to stomatin satisfies a needin the art by providing new diagnostic or therapeutic compositionsuseful in the treatment of disorders associated with abnormal iontransport or membrane conductance.

SUMMARY OF THE INVENTION

[0008] The present invention features a novel integral membrane proteinhereinafter designated IMP and characterized as having similarity tohuman stomatin, the Caenorhabditis elegans proteins MEC-2 and UNC-24, aMycobacterium tuberculosis protein, and a membrane protein fromMethanococcus jannaschii.

[0009] Accordingly, the invention features a substantially purifiedpolypeptide having the amino acid sequence shown in SEQ ID No: 1 orfragments thereof. Preferred fragments of SEQ ID No: 1 are fragments ofabout 15 amino acids or greater in length which define fragments unique(i.e., having less than about 25% identity to fragments of anotherprotein) to SEQ ID No: 1 or which retain biological activity (e.g.,regulation of ion channel activity) or immunological activity (i.e.,capable of eliciting anti-IMP antibodies).

[0010] The present invention further provides isolated and substantiallypurified polynucleotide sequences encoding the polypeptide comprisingthe amino acid sequence of SEQ ID No: 1 or fragments thereof. In aparticular aspect, the polynucleotide is the nucleotide sequence of SEQID No: 2 or variants thereof. In another embodiment, the presentinvention provides polynucleotides comprising fragments of SEQ ID No: 2having a length greater than 30 nucleotides. The invention furthercontemplates fragments of this polynucleotide sequence (i.e., SEQ ID No:2) that are at least 50 nucleotides, at least 100 nucleotides, at least250 nucleotides, and at least 500 nucleotides in length.

[0011] In addition, the invention provides polynucleotide sequenceswhich hybridize under stringent conditions to the polynucleotidesequence of SEQ ID No: 2. In another embodiment the present inventionprovides a composition comprising an isolated and purifiedpolynucleotide sequence encoding IMP.

[0012] The invention provides polynucleotide sequences comprising thecomplement of SEQ ID No: 2 or variants thereof; these complementarynucleic acid sequences may comprise the complement of the entire nucleicacid sequence of SEQ ID No: 2 or fragments thereof. In anotherembodiment the present invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID No: 2 or variants thereof.

[0013] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonicleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode IMP.

[0014] In another embodiment, the present invention provides an isolatedpolynucleotide comprising at least a portion of the nucleic acidsequence of SEQ ID No: 2 or variants thereof contained on a recombinantexpression vector. In yet another embodiment, the expression vectorcontaining the polynucleotide sequence is contained within a host cell.The invention is not limited by the nature of the host cell employed.For example, the host cell may be an E. coli cell, a yeast cell, aninsect cell, a mammalian cell, etc.

[0015] The present invention also provides a method for producing apolypeptide comprising the amino acid sequence of SEQ ID No: 1 orfragments thereof, the method comprising the steps of: a) culturing thehost cell containing an expression vector containing an isolatedpolynucleotide encoding at least a fragment of the IMP polypeptide underconditions suitable for the expression of the polypeptide; and b)recovering the polypeptide from the host cell culture.

[0016] In another embodiment, the invention provides a pharmaceuticalcomposition comprising a substantially purified human IMP protein havingan amino acid sequence of SEQ ID No: 1 in conjunction with a suitablepharmaceutical carrier.

[0017] The invention also provides a purified antibody which bindsspecifically to a polypeptide comprising at least a portion of the aminoacid sequence of SEQ ID No: 1.

[0018] Still further, the invention provides a purified agonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ IDNo: 1. The present invention further provides a pharmaceuticalcomposition comprising a purified agonist which specifically binds toand modulates the activity of a polypeptide comprising at least aportion of the amino acid sequence of SEQ ID No: 1. In anotherembodiment, the invention provides a purified antagonist whichspecifically binds to and modulates the activity of a polypeptidecomprising at least a portion of the amino acid sequence of SEQ IDNo: 1. The present invention further provides a pharmaceuticalcomposition comprising a purified antagonist which specifically binds toand modulates the activity of a polypeptide comprising at least aportion of the amino acid sequence of SEQ ID No: 1.

[0019] The invention also provides a method for treating prostate cancercomprising administering to a subject in need of such treatment aneffective amount of a pharmaceutical composition comprising a purifiedantagonist which specifically binds to and modulates the activity of apolypeptide comprising at least a portion of the amino acid sequence ofSEQ ID No: 1.

[0020] The invention also provides a method for detection ofpolynucleotides encoding human IMP in a biological sample comprising thesteps of: a) hybridizing a polynucleotide sequence encoding human IMP(SEQ ID No: 1) to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding human IMP in the biologicalsample. In a preferred embodiment, prior to hybridization, the nucleicacid material of the biological sample is amplified by the polymerasechain reaction.

BRIEF DESCRIPTION OF THE FIGURES

[0021]FIGS. 1A, 1B, 1C and 1D shows the amino acid sequence (SEQ IDNo: 1) and nucleic acid sequence (SEQ ID No: 2) of IMP. The alignmentwas produced using MacDNASIS PRO™ software (Hitachi Software EngineeringCo., Ltd., San Bruno, Calif.).

[0022]FIG. 2A, 2B and 2C shows the amino acid sequence alignments amongIMP (SEQ ID No: 1), human erythrocyte band 7 protein (i.e., stomatin)[GI 31069 (SEQ ID No: 3); Hiebl-Dirschmied, C. M. et al. (1991) Biochem.Biophys. Acta 1090:123-24], C. elegans MEC-2 protein [GI 1065452 (SEQ IDNo: 4); Huang, M. (1995), supra], C. elegans UNC-24 protein [GI 1353669(SEQ ID No: 5); Barnes, T. M. (1996), supra], a Mycobacteriumtuberculosis protein of unknown function [Z79701 (SEQ ID No: 6)] and amembrane protein from Methanococcus jannaschii [GI 1591514 (SEQ ID No:7); Bult, C. J. et al. (1996) Science 273:1058-731 . The alignment wasproduced using the multisequence alignment program of DNASTAR™ software(DNASTAR Inc, Madison Wis.).

[0023]FIG. 3 shows the northern analysis for SEQ ID No: 2. The northernanalysis was produced electronically using LIFESEQ™ database (IncytePharmaceuticals, Inc., Palo Alto, Calif.).

DESCRIPTION OF THE INVENTION

[0024] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0025] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0026] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

[0027] “Nucleic acid sequence” as used herein refers to anohgonucleotide, nucleotide, or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represents the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0028] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encoding IMP(SEQ ID No: 1) or fragments thereof (e.g., SEQ ID No: 2 and fragmentsthereof) may be employed as hybridization probes. In this case, theIMP-encoding polynucleotide sequences are typically employed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS)and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

[0029] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0030] “Peptide nucleic acid', as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0031] IMP, as used herein, refers to the amino acid sequences ofsubstantially purified IMP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0032] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0033] A “variant” of IMP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0034] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0035] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0036] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0037] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic IMP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0038] The term “agonist”, as used herein, refers to a molecule which,when bound to IMP, causes a change in IMP which modulates the activityof IMP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to IMP.

[0039] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to IMP, blocks or modulates the biological orimmunological activity of IMP. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to IMP.

[0040] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of IMP. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of IMP.

[0041] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of IMPor portions thereof and, as such, is able to effect some or all of theactions of stomatin-like molecules.

[0042] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding IMP or the encoded IMP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0043] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0044] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0045] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0046] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0047] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0048] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0049] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0050] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences. Under “stringentconditions” SEQ ID No: 2 or fragments thereof will hybridize to itsexact complement and closely related sequences. The stringent conditionsare chosen such that SEQ ID No: 2 or fragments thereof will hybridize tosequences encoding human IMP, but not to sequences encoding humanstomatin (GI 31068) or the M. tuberculosis protein (see GI 1524225).When fragments of SEQ ID No: 2 are employed in hybridization reactions,the stringent conditions include the choice of fragments of SEQ ID No: 2to be used, unique sequences or regions which are either non-homologousto or which contain less than about 50% homology or complementarity withthe nucleotides encoding human stomatin (GI 30168) or the M.tuberculosis protein (see GI 1524225).

[0051] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0052] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ ID No:1” encompasses the full-length human IMP and fragments thereof.

[0053] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0054] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0055] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0056] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding IMP orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0057] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID No: 2 by northern analysis is indicativeof the presence of mRNA encoding IMP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0058] “Alterations” in the polynucleotide of SEQ ID No: 2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding IMP including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes IMP (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID No: 2),the inability of a selected fragment of SEQ ID No: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding IMP (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosome spreads).

[0059] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind IMPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0060] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

[0061] The invention is based on the discovery of a novel human integralmembrane protein (IMP), the polynucleotides encoding IMP, and the use ofthese compositions for the diagnosis, prevention, or treatment ofdiseases associated with abnormal ion transport or membrane conductance.In addition, as mRNA encoding IMP is found in a number of tumors, IMPserves as a marker for cancerous cells, particularly prostate tumorcells.

[0062] Nucleic acids encoding the human IMP of the present inventionwere first identified in Incyte Clone 789094 from the PROSTUT03 cDNAlibrary through a computer-generated search for amino acid sequencealignments. A consensus sequence, SEQ ID No: 2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 220581 (STOMNOT01), 968553 (BRSTNOT05), 748425 (BRAITUT01),789094 (PROSTUT03), 604240 (BRSTTUT01), 1556986 (BLADTUT04) and 323059(EOSIHET02).

[0063] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID No: 1, as shown in FIG. 1Aand 1B. IMP is 351 amino acids in length and contains two cysteineresidues (C₁₆₇, and C₁₇₂). In addition to providing sites for disulfidebond formation, the cysteine residues provide potential sites forpalmitoylation; the stomatin protein, which shares homology with IMP,has been shown to be palmitoylated [Wang, D et al. (1991) J. Biol. Chem.266:17826]. The human IMP of the present invention contains numerouspotential O-linked glycosylation sites (i.e., serine and threonineresidues). IMP has two potential N-linked glycoslyation sites (i.e.,Asn-X-Ser/Thr) (i.e., N₉₆ and N₁₅₄). In addition, the human IMP of thepresent invention contains numerous potential phosphorylation sites(i.e., typically the hydroxyl groups of serine, threonine and tyrosineresidues although asparagine, histidine and lysine residues may also bephosphorylated), including potential sites for phosphorylation bycAMP-dependent protein kinase (e.g., R-X-S/T) (i.e., S₂₉, T₃₆, S₁₅₂ andS₂₁₃).

[0064] The IMP protein of the present invention, like C. elegans UNC-24,has an acidic isoelectric point (pI). IMP has a pI of 5.91, and UNC-24has a pI of 5.11.

[0065] IMP has chemical and structural homology with stomatin (GI 31069;SEQ ID No: 3), (GI 31069; SEQ ID No: 3), C. elegans MEC-2 protein (GI1065452; SEQ ID No: 4), C. elegans UNC-24 protein (GI 1353669; SEQ IDNo: 5), a Mycobacterium tuberculosis protein of unknown function(Z79701; SEQ ID No: 6) and a membrane protein from Methanococcusjannaschii (GI 1591514; SEQ ID No: 7). In particular, residues 79-209 ofIMP are strongly sirnilar to residues 96-226 of stomatin (33% identity,60% similarity) and residues 218-253 of IMP are strongly similar toresidues 211-246 of stomatin (30% identity, 52% similarity). Residues79- 214 of IMP are strongly similar to residues 101-236 of C. elegansMEC-2 (35% identity and 62% similarity). Residues 37-75, 45-71, 83-132,107-134 and 263-283 of IMP are strongly similar to residues 88-126,95-121,133-181, 156-183, and 307-326, respectively of C. elegans UNC-24(34% identity and 61% similarity; 41% identity and 63% similarity; 34%identity and 62% similarity; 43% identity and 57% similarity; and 25%identity and 70% similarity, respectively). Residues 38-262 and 187-265of IMP are strongly similar to residues 26-250 and 186-264, respectivelyof the M. tuberculosis protein (Z79701) (38% identity and 61%similarity; and 36% identity and 63% similarity, respectively). Residues38-71, 83-211 and 202-222 of IMP are strongly similar to residues 25-58,69-197 and 177-197, respectively of the M. jannaschii membrane protein(GI 1591514) (44% identity and 67% similarity; 40% identity and 67%similarity; and 38% identity and 66% similarity, respectively). A pairof residues are said to be similar if they represent conservativesubstitutions. FIG. 2A, 2B and 2C provides an alignment between theamino acid sequences of SEQ ID NOs:1 and 3-7.

[0066] Northern analysis (FIG. 3) shows the expression of this sequencein various libraries, at least 38% of which are cancerous orimmortalized. Of particular note is the expression of IMP mRNA inprostate tumor ({fraction (2/13)}), breast tumor ({fraction (1/13)}),and pancreatic tumor libraries ({fraction (1/13)}). This pattern ofexpression demonstrates that IMP serves as a marker for cancerous cells,particularly prostate tumor cells.

[0067] The invention also encompasses IMP variants. A preferred IMPvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the IMP amino acid sequence (SEQ ID No: 1). Amost preferred IMP variant is one having at least 95% amino acidsequence similarity to SEQ ID No: 1.

[0068] The invention also encompasses polynucleotides which encode IMP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of IMP can be used to generate recombinant molecules whichexpress IMP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID No: 2 asshown in FIG. 1A and 1B.

[0069] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding IMP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring IMP, and all such variations are to beconsidered as being specifically disclosed.

[0070] Although nucleotide sequences which encode IMP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring IMP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding IMP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding IMP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0071] The invention also encompasses production of DNA sequences, orportions thereof, which encode IMP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding IMP or any portion thereof.

[0072] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID No: 2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0073] Altered nucleic acid sequences encoding IMP which are encompassedby the invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent IMP. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentIMP. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of IMP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and argimine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

[0074] Also included within the scope of the present invention arealleles of the genes encoding IMP. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0075] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0076] The nucleic acid sequences encoding IMP may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0077] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0078] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111 - 119). In this method, multiple restrictionenzyme digestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performiing PCR.

[0079] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions. When screening for full-length cDNAs, it ispreferable to use libraries that have been size-selected to includelarger cDNAs. Also, random-primed libraries are preferable, in that theywill contain more sequences which contain the 5′ regions of genes. Useof a randomly primed library may be especially preferable for situationsin which an oligo d(T) library does not yield a full-length cDNA.Genomic libraries may be useful for extension of sequence into the 5′and 3′ non-transcribed regulatory regions.

[0080] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. Genotyper™ andSequence Navigator™, Perkin Elmer) and the entire process from loadingof samples to computer analysis and electronic data display may becomputer controlled. Capillary electrophoresis is especially preferablefor the sequencing of small pieces of DNA which might be present inlimited amounts in a particular sample.

[0081] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode IMP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of IMP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressIMP.

[0082] As will be understood by those of skill in the art, it may beadvantageous to produce IMP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0083] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterIMP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0084] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding IMP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of IMP activity, it may be useful toencode a chimeric IMP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the IMP encoding sequence and theheterologous protein sequence, so that IMP may be cleaved and purifiedaway from the heterologous moiety.

[0085] In another embodiment, sequences encoding IMP may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of IMP, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0086] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of IMP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0087] In order to express a biologically active IMP, the nucleotidesequences encoding IMP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0088] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding IMPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0089] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding IMP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0090] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding IMP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0091] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for IMP. For example, whenlarge quantities of IMP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding IMP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wiss.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutatlione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0092] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544. In caseswhere plant expression vectors are used, the expression of sequencesencoding IMP may be driven by any of a number of promoters. For example,viral promoters such as the 35S and 19S promoters of CaMV may be usedalone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0093] An insect system may also be used to express IMP. For example, inone such system, Autographa californica nuclear polyhedrosis virus(ACNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding IMPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of IMP will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which IMP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0094] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding IMP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing IMP in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in marmalian host cells.

[0095] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding IMP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding IMP, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0096] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the mserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0097] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress IMP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0098] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, B glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0099] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding IMP isinserted within a marker gene sequence, recombinant cells containingsequences encoding IMP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding IMP under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0100] Alternatively, host cells which contain the nucleic acid sequenceencoding IMP and express IMP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0101] The presence of polynucleotide sequences encoding IMP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding IMP. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding IMP to detect transformantscontaining DNA or RNA encoding IMP. As used herein “olgonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

[0102] A variety of protocols for detecting and measuring the expressionof IMP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson IMP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0103] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding IMPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding IMP, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland,Ohio). Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

[0104] Host cells transformed with nucleotide sequences encoding IMP maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeIMP may be designed to contain signal sequences which direct secretionof IMP through a prokaryotic or eukaryotic cell membrane. When it isdesired to express a secreted form of IMP, a polynucleotide sequenceencoding a portion of the IMP lacking the hydrophobic stretches locatedat residues 29-44 and 58-70 of SEQ ID No: 1 (either or both of thesestretches may anchor IMP in the membrane) is preferentially employed.

[0105] Other recombinant constructions may be used to join sequencesencoding IMP to nucleotide sequence encoding a polypeptide domain whichwill facilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and IMP may be used to facilitate purification. Onesuch expression vector provides for expression of a fusion proteincontaining IMP and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying IMP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0106] In addition to recombinant production, fragments of IMP may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of IMP may bechemically synthesized separately and combined using chemical methods toproduce the fall length molecule.

THERAPEUTICS

[0107] Based on the chemical and structural homology among IMP (SEQ IDNo: 1) and stomatin (SEQ ID No: 3), and stomatin-like proteins [e.g.,MEC-2 (SEQ ID No: 4), UNC-24 (SEQ ID No: 5), the M. tuberculosis protein(Z79701; SEQ ID No: 6) and M. jannaschii membrane protein (GI 1591514;SEQ ID No: 7), IMP appears to play a role in the regulation of ionchannels. Improper functioning of ion channels is associated with avariety of disorders including, but not limited to, hemolytic anemias(e.g., hereditary stomatocytosis, hydrocytosis and xerocytosis).Furthermore, since the sequences encoding IMP were isolated fromprostate tumor tissue and IMP is also expressed in breast and pancreatictumors, IMP expression appears to be indicative of a proliferative cellstate.

[0108] Therefore, in one embodiment, IMP or a fragment or derivativethereof may be administered to a subject to treat disorders associatedwith abnormal ion transport or membrane conductance as well as a varietyof tumors. Such conditions and diseases may include, but are not limitedto, hemolytic anemias and prostate, breast and pancreatic tumors.

[0109] In another embodiment, a vector capable of expressing IMP, or afragment or a derivative thereof, may also be administered to a subjectto treat the hemolytic anemias and prostate, breast and pancreatictumors described above.

[0110] In one aspect, agonists of IMP may be used to increase theactivity of IMP in cells having reduced IMP-associated ion transport.

[0111] In one embodiment, antagonists or inhibitors of IMP may beadministered to a subject to treat or prevent tumors, particularlyprostate, breast and pancreatic tumors.

[0112] In another embodiment, a vector expressing antisense of thepolynucleotide encoding IMP may be administered to a subject to treat orprevent tumors, particularly prostate, breast and pancreatic tumors.

[0113] In other embodiments, IMP may be administered in combination withother conventional chemotherapeutic agents. The combination oftherapeutic agents having different mechanisms of action will havesynergystic effects allowing for the use of lower effective doses ofeach agent and lessening side effects.

[0114] Antagonists or inhibitors of IMP may be produced using methodswhich are generally known in the art. In particular, purified IMP may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind IMP.

[0115] Antibodies which are specific for IMP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express IMP.The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which reduce or abolish IMP activity) are especially preferred fortherapeutic use.

[0116] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith IMP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0117] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to IMP have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of IMP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0118] Monoclonal antibodies to IMP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al.(1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984)Mol. Cell Biol. 62:109-120).

[0119] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceIMP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0120] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0121] Antibody fragments which contain specific binding sites for IMPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0122] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between IMP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering IMP epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0123] In another embodiment of the invention, the polynucleotidesencoding IMP, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding IMP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding IMP. Thus, antisense molecules may be used to modulate IMPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding IMP.

[0124] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encoding IMP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0125] Genes encoding IMP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes IMP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0126] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding IMP, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0127] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding IMP.

[0128] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0129] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding IMP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0130] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0131] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0132] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0133] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of IMP,antibodies to IMP, mimetics, agonists, antagonists, or inhibitors ofIMP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0134] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0135] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0136] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0137] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0138] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0139] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0140] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0141] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0142] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0143] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0144] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of IMP, such labeling wouldinclude amount, frequency, and method of administration.

[0145] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0146] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0147] A therapeutically effective dose refers to that amount of activeingredient, for example IMP or fragments thereof, antibodies of IMP,agonists, antagonists or inhibitors of IMP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0148] Pharmaceutical compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies is used in formulating a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0149] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0150] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

DIAGNOSTICS

[0151] In another embodiment, antibodies which specifically bind IMP maybe used for the diagnosis of conditions or diseases characterized byexpression of IMP, or in assays to monitor patients being treated withIMP, agonists, antagonists or inhibitors. The antibodies useftil fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for IMP includemethods which utilize the antibody and a label to detect IMP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0152] A variety of protocols including ELISA, RIA, and FACS formeasuring IMP are known in the art and provide a basis for diagnosingaltered or abnormal levels of IMP expression. Normal or standard valuesfor IMP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to IMP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of IMPexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0153] In another embodiment of the invention, the polynucleotidesencoding IMP is used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofIMP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of IMP, andto monitor regulation of IMP levels during therapeutic intervention.

[0154] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding IMP or closely related molecules, may be used to identifynucleic acid sequences which encode IMP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding IMP, alleles, or related sequences.

[0155] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the IMP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID No: 2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring IMP.

[0156] Means for producing specific hybridization probes for DNAsencoding IMP include the cloning of nucleic acid sequences encoding IMPor IMP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0157] Polynucleotide sequences encoding IMP may be used for thediagnosis of conditions or diseases which are associated with expressionof IMP. Examples of such conditions or diseases include cancers of theprostate, pancreas, and breast as well as disorders associated withaltered ion conductance. The polynucleotide sequences encoding IMP maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dip stick, pin,ELISA or chip assays utilizing fluids or tissues from patient biopsiesto detect altered IMP expression. Such qualitative or quantitativemethods are well known in the art.

[0158] In a particular aspect, the nucleotide sequences encoding IMPprovide the basis for assays that detect activation or induction ofvarious cancers, particularly those mentioned above; in addition thelack of expression of IMP may be detected using the IMP-encodingnucleotide sequences disclosed herein. The nucleotide sequences encodingIMP may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding IMP in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

[0159] In order to provide a basis for the diagnosis of diseaseassociated with expression of IMP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes IMP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0160] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0161] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0162] Additional diagnostic uses for oligonucleotides designed from thesequences encoding IMP may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′−>3′) and another withantisense (3′<−5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0163] Methods which may also be used to quantitate the expression ofIMP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

[0164] In another embodiment of the invention, the nucleic acidsequences which encode IMP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques . Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127- 134, and Trask, B.J.(1991) Trends Genet. 7:149-154.

[0165] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265: 198 1f). Correlation between the location of thegene encoding IMP on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0166] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0167] In another embodiment of the invention, IMP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between IMPand the agent being tested, may be measured.

[0168] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to IMP large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with IMP, or fragments thereof, and washed. Bound IMP is thendetected by methods well known in the art. Purified IMP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0169] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding IMPspecifically compete with a test compound for binding IMP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with IMP.

[0170] In additional embodiments, the nucleotide sequences which encodeIMP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0171] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0172] I PROSTUT03 cDNA Library Construction

[0173] The PROSTUT03 cDNA library was constructed from prostate tumortissue removed from a 76-year-old Caucasian male by radicalprostatectomy. The pathology report indicated Mayo grade 3 (of 4)adenocarcinoma (Gleason grade 3+3) in the periphery of the prostate.Perineural invasion was present as was involvement of periprostatictissue. Non-tumorous portions of the prostate exhibited adenofibromatoushyperplasia. The patient had elevated levels of prostate specificantigen (PSA). Pelvic lymph nodes were negative for tumor. A priorstomach ulcer and atherosclerosis were reported in the patient history;however, the patient was not on any medication at the time of surgery.

[0174] The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron-PT 3000 (Brinkmann Instruments, Inc. Westbury N.Y.)in guanidinium isothiocyanate solution. The lysate was extracted twicewith acid phenol at pH 4.0, the first time per Stratagene's RNAisolation protocol (Stratagene Inc, San Diego Calif.). The RNA wasprecipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in DEPC-treated water, and DNase treated for 25 min at 37°C. The RNA was re-extracted as described above, and mRNA was isolatedusing the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth Calif.) and usedto construct the cDNA library.

[0175] The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(catalog #18248-013; Gibco/BRL). cDNAs were fractionated on a SepharoseCL4B column (catalog #275105, Pharmacia), and those cDNAs exceeding 400bp were ligated into pSport I. The plasmid pSport I was subsequentlytransformed into DH5a™ competent cells (Cat. #18258-012, Gibco/BRL).

[0176] II Isolation and Sequencing of cDNA Clones

[0177] Plasmid DNA was released from the cells and purified using theMiniprep Kit (Catalog #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #2271 1, LIFE TECHNOLOGIES™,Gaithersburg Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2)the bacteria were cultured for 24 hours after the wells were inoculatedand then lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R rotor at 2900 rpm for 5 minutes wasperformed before the contents of the block were added to the primaryfilter plate; and 4) the optional step of adding isopropanol to TRISbuffer was not routinely performed. After the last step in the protocol,samples were transferred to a Beckman 96-well block for storage.

[0178] The cDNAs were sequenced by the method of Sanger F and AR Coulson(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems, and the reading frame was determined.

[0179] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0180] Each cDNA was compared to sequences in GenBank using a searchalgorithm developed by Applied Biosystems and incorporated into theINHERIT™ 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles, Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

[0181] Peptide and protein sequence homologies were ascertained usingthe INHERIT-670 sequence analysis system using the methods similar tothose used in DNA sequence homologies. Pattern Specification Languageand parameter windows were used to search protein databases forsequences containing regions of homology which were scored with aninitial value. Dot-matrix homology plots were examined to distinguishregions of significant homology from chance matches.

[0182] BLAST, which stands for Basic Local Alignment Search Tool(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990)J. Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amio acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

[0183] An HSP consists of two sequence fragments of arbitrary but equallengths whose alignment is locally maximal and for which the alignmentscore meets or exceeds a threshold or cutoff score set by the user. TheBLAST approach is to look for HSPs between a query sequence and adatabase sequence, to evaluate the statistical significance of anymatches found, and to report only those matches which satisfy theuser-selected threshold of significance. The parameter E establishes thestatistically significant threshold for reporting database sequencematches. E is interpreted as the upper bound of the expected frequencyof chance occurrence of an HSP (or set of HSPs) within the context ofthe entire database search. Any database sequence whose match satisfiesE is reported in the program output.

[0184] A comparison of the full-length and partial cDNA sequences andthe deduced amino acid sequences corresponding to the human IMP gene andIMP protein with known nucleotide and protein sequences in GenBankrevealed that the full-length human IMP cDNA and protein sequences(i.e., SEQ ID NOS:1 and 2) were unique (i.e., not previouslyidentified). This search revealed that the human IMP protein shared somehomology with human stomatin (SEQ ID No: 3), C. elegans MEC-2 (SEQ IDNo: 4), C. elegans UNC-24 (SEQ ID No: 5), a protein from M. tuberculosis(SEQ ID No: 6) and a membrane protein from M. jannaschii (SEQ ID No: 7)(see alignment in FIG. 2A, 2B, and 2C).

[0185] IV Northern Analysis

[0186] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0187] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0188] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0189] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0190] The results of northern analysis are reported as a list oflibraries in which the transcript encoding IMP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0191] Electronic northern analysis (FIG. 3) revealed that mRNA encodinghuman IMP (SEQ ID No: 1) was present in libraries generated from thefollowing tissues: colon, testis, liver, heart, keratinocytes, brain,lung and paraganglia. The expression of IMP in such a wide variety oftissues is similar to the ubiquitous express of stomatin [Stewart, G. W.et al. (1992), supra]. In addition to expression in apparently normalhuman tissues, IMP was expressed in tumors of the prostate, pancreas andbreast as well as in an immortalized cell line.

[0192] V Extension of IMP-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0193] Full length IMP-encoding nucleic acid sequence (SEQ ID No: 2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0194] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0195] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

[0196] Step 1 94° C. for 1 min (initial denaturation)

[0197] Step 2 65° C. for 1 min

[0198] Step 3 68° C. for 6 min

[0199] Step 4 94° C. for 15 sec

[0200] Step 5 65° C. for 1 min

[0201] Step 6 68° C. for 7 min

[0202] Step 7 Repeat step 4-6 for 15 additional cycles

[0203] Step 8 94° C. for 15 sec

[0204] Step 9 65° C. for 1 min

[0205] Step 10 68° C. for 7:15 min

[0206] Step 11 Repeat step 8-10 for 12 cycles

[0207] Step 12 72° C. for 8 min

[0208] Step 13 4° C. (and holding)

[0209] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0210] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μT4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

[0211] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions:

[0212] Step 1 94° C. for 60 sec

[0213] Step 2 94° C. for 20 sec

[0214] Step 3 55° C. for 30 sec

[0215] Step 4 72° C. for 90 sec

[0216] Step 5 Repeat steps 2-4 for an additional 29 cycles

[0217] Step 6 72° C. for 180 sec

[0218] Step 7 4° C. (and holding)

[0219] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0220] VI Labeling and Use of Hybridization Probes

[0221] Hybridization probes derived from SEQ ID No: 2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

[0222] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N H). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0223] VII Antisense Molecules

[0224] Antisense molecules to the IMP-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring IMP. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of IMP, as shown in FIG. 1A and 1B, is used toinhibit expression of naturally occurring IMP. The complementaryoligonucleotide is designed from the most unique 5′ sequence as shown inFIG. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an IMP-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5′ sequence of SEQ ID No:2, an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5′ codingsequence of the polypeptide as shown in FIG. 1A and 1B.

[0225] VIII Expression of IMP

[0226] Expression of IMP is accomplished by subloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express IMP in E. coli .Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0227] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of 13-galactosidase, about 5 to 15 residues oflinker, and the full length protein or fragments thereof. Sequencesencoding IMP fusion proteins lacking the hydrophobic stretches locatedat residues 29-44 and 58-70 of SEQ ID No: 1 (either or both of thesestretches may anchor IMP in the membrane) are preferentially employedfor the production of soluble forms of recombinant IMP. The signalresidues present on the pSport vector direct the secretion of IMP intothe bacterial growth media which can be used directly in the followingassay for activity.

[0228] Alternatively, IMP may be expressed as a membrane-bound proteinin a host cell and the recombinant IMP recovered from the membrane ofthe host cell using techniques well known to the art [e.g., Stewart, G.W. (1992), supra].

[0229] IX Demonstration of IMP Activity

[0230] Given the chemical and structural similarity between IMP andstomatin and stomatin-like proteins (e.g., MEC-2 and UNC-24), IMP ispresumed to act as a regulator of an ion channel. To demonstrate IMP'sability to regulate ion channels, cells which normally express IMP[e.g., the HUV-EC-C endothelial cell line (ATCC CRL 1730)] are madedeficient in IMP expression (or deficient in IMP activity) and theeffect of reduced or absent IMP expression/activity upon the transportof ions is examined. Several methods for the measurement ofintracellular ion concentration (e.g., Na⁺ and K⁺) and the measurementof ion flux are known to the art [see, e.g., Stewart, G. W. et al.(1992), supra, Stewart, G. W. (1988) J. Physiol. (Loud) 401:1, andStein, W. D. (1990) Channels, Carriers and Pumps, Acad. Press, London].

[0231] The expression of IMP may be reduced or abolished by introductionof a sufficient amount of antisense IMP molecules. Antisense transcriptsmay be introduced by transformation of the IMP-expressing cell with anexpression vector which produced antisense IMP transcripts oralternatively, antisense IMP transcripts may be chemically synthesizedand introduced (e.g., via microinjection, electroporation, liposomefusion) into the IMP-expressing cell. The activity of IMP in anIMP-expressing cell may be reduced or abolished by the introduction ofanti-IMP antibodies capable of neutralizing the activity of IMP.

[0232] Following the introduction of either anti-IMP antibodies orantisense IMP transcripts to generate modified cells, the ability of thecell to maintain proper ion conductance is examined using standardtechniques. Failure of the modified cells to maintain proper ionconductance indicates that IMP regulates an ion channel. This is furtherdemonstrated by restoration of proper ion conductance by the addition ofpurified IMP (prepared as described in Ex. VIII) to the modified cells(purified IMP may be introduced into the modified cells by a variety ofmeans including direct injection, electroporation, liposome fusion,etc.).

[0233] X Production of IMP Specific Antibodies

[0234] IMP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID No: 2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0235] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0236] XI Purification of Naturally Occurring IMP Using SpecificAntibodies

[0237] Naturally occurring or recombinant IMP is substantially purifiedby immunoaffinity chromatography using antibodies specific for IMP. Animmunoaffinity column is constructed by covalently coupling IMP antibodyto an activated chromatographic resin, such as CnBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0238] Media containing IMP is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of IMP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/IMP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and IMP is collected.

[0239] XII Identification of Molecules Which Interact with IMP

[0240] IMP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled IMP, washed and any wells withlabeled IMP complex are assayed. Data obtained using differentconcentrations of IMP are used to calculate values for the number,affinity, and association of IMP with the candidate molecules.

[0241] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 7 356 amino acids amino acid single linear Consensus Consensus 1 MetLeu Ala Arg Ala Ala Arg Gly His Trp Gly Pro Phe Ala Glu Gly 1 5 10 15Leu Ser Thr Gly Phe Trp Pro Arg Ser Gly Arg Ala Ser Ser Gly Leu 20 25 30Pro Arg Asn Thr Val Val Leu Phe Val Pro Gln Gln Glu Ala Trp Val 35 40 45Val Glu Arg Met Gly Arg Phe His Arg Ile Leu Glu Pro Gly Leu Asn 50 55 60Ile Leu Ile Pro Val Leu Asp Arg Ile Arg Tyr Val Gln Ser Leu Lys 65 70 7580 Glu Ile Val Ile Asn Val Pro Glu Gln Ser Ala Val Thr Leu Asp Asn 85 9095 Val Thr Leu Gln Ile Asp Gly Val Leu Tyr Leu Arg Ile Met Asp Pro 100105 110 Tyr Lys Ala Ser Tyr Gly Val Glu Asp Pro Glu Tyr Ala Val Thr Gln115 120 125 Leu Ala Gln Thr Thr Met Arg Ser Glu Leu Gly Lys Leu Ser XaaAsp 130 135 140 Lys Val Phe Arg Glu Arg Glu Ser Leu Asn Ala Ser Ile ValAsp Ala 145 150 155 160 Ile Asn Gln Ala Ala Asp Cys Trp Gly Ile Arg CysLeu Arg Tyr Glu 165 170 175 Ile Lys Asp Ile His Val Pro Pro Arg Val LysGlu Ser Met Gln Met 180 185 190 Gln Val Glu Ala Glu Arg Arg Lys Arg AlaThr Val Leu Glu Ser Glu 195 200 205 Gly Thr Arg Glu Ser Ala Ile Asn ValAla Glu Gly Lys Lys Gln Ala 210 215 220 Gln Ile Leu Ala Ser Glu Ala GluLys Ala Glu Gln Ile Asn Gln Ala 225 230 235 240 Ala Gly Glu Ala Ser AlaVal Leu Ala Lys Ala Lys Ala Lys Ala Glu 245 250 255 Ala Ile Arg Ile LeuAla Ala Ala Leu Thr Gln His Asn Gly Asp Ala 260 265 270 Ala Ala Ser LeuThr Val Ala Glu Gln Tyr Val Ser Ala Phe Ser Lys 275 280 285 Leu Ala LysAsp Ser Asn Thr Ile Leu Leu Pro Ser Asn Pro Gly Asp 290 295 300 Val ThrSer Met Val Ala Gln Ala Met Gly Val Tyr Gly Ala Leu Thr 305 310 315 320Lys Ala Pro Val Pro Gly Thr Pro Asp Ser Leu Ser Ser Gly Ser Ser 325 330335 Arg Asp Val Gln Gly Thr Asp Ala Ser Xaa Asp Glu Glu Leu Asp Arg 340345 350 Val Lys Met Ser 355 1188 base pairs nucleic acid single linearConsensus Consensus 2 GGCTTCTGGG AGCNACCGCT CCGCTCGTCT CGTTGGTTCCGGAGGTCGCT GCGGCGGTGG 60 GAAATGCTGG CGCGCGCGGC GCGGGGGCAC TGGGGCCCTTTTGCTGAGGG GCTCTCTACT 120 GGCTTCTGGC CGCGCTCCGG CCGCGCCTCC TCTGGATTGCCCCGAAACAC CGTGGTACTG 180 TTCGTGCCGC AGCAGGAGGC CTGGGTGGTG GAGCGAATGGGCCGATTCCA CCGGATCCTG 240 GAGCCTGGTT TGAACATCCT CATCCCTGTG TTAGACCGGATCCGATATGT GCAGAGTCTC 300 AAGGAAATTG TCATCAACGT GCCTGAGCAG TCGGCTGTGACTCTCGACAA TGTAACTCTG 360 CAAATCGATG GAGTCCTTTA CCTGCGCATC ATGGACCCTTACAAGGCAAG CTACGGTGTG 420 GAGGACCCTG AGTATGCCGT CACCCAGCTA GCTCAAACAACCATGAGATC AGAGCTCGGC 480 AAACTCTCTN TGGACAAAGT CTTCCGGGAA CGGGAGTCCCTGAATGCCAG CATTGTGGAT 540 GCCATCAACC AAGCTGCTGA CTGCTGGGGT ATCCGCTGCCTNCGTTATGA GATCAAGGAT 600 ATCCATGTGC CACCCCGGGT GAAAGAGTCT ATGCAGATGCAGGTGGAGGC AGAGCGGCGG 660 AAACGGGCCA CAGTTCTAGA GTCTGAGGGG ACCCGAGAGTCGGCCATCAA TGTGGCAGAA 720 GGGAAGAAAC AGGCCCAGAT CCTGGCCTCC GAAGCAGAAAAGGCTGAACA GATAAATCAG 780 GCAGCAGGAG AGGCCAGTGC AGTTCTGGCG AAGGCCAAGGCTAAAGCTGA AGCTATTCGA 840 ATCCTGGCTG CAGCTCTGAC ACAACATAAT GGAGATGCAGCAGCTTCACT GACTGTGGCC 900 GAGCAGTATG TCAGCGCGTT CTCCAAACTG GCCAAGGACTCCAACACTAT CCTACTGCCC 960 TCCAACCCTG GCGATGTCAC CAGCATGGTG GCTCAGGCCATGGGTGTATA TGGAGCCCTC 1020 ACCAAAGCCC CAGTGCCAGG GACTCCAGAC TCACTCTCCAGTGGGAGCAG CAGAGATGTC 1080 CAGGGTACAG ATGCAAGTNT TGATGAGGAA CTTGATCGAGTCAAGATGAG TTAGTGGAGC 1140 TGGGCTTNGC CAGGGAGTCT GGGGACAAGG AAGCAGATTTTCCTGATT 1188 288 amino acids amino acid single linear Genbank 31069 3Met Ala Glu Lys Arg His Thr Arg Asp Ser Glu Ala Gln Arg Leu Pro 1 5 1015 Asp Ser Phe Lys Asp Ser Pro Ser Lys Gly Leu Gly Pro Cys Gly Trp 20 2530 Ile Leu Val Ala Phe Ser Phe Leu Phe Thr Val Ile Thr Phe Pro Ile 35 4045 Ser Ile Trp Met Cys Ile Lys Ile Ile Lys Glu Tyr Glu Arg Ala Ile 50 5560 Ile Phe Arg Leu Gly Arg Ile Leu Gln Gly Gly Ala Lys Gly Pro Gly 65 7075 80 Leu Phe Phe Ile Leu Pro Cys Thr Asp Ser Phe Ile Lys Val Asp Met 8590 95 Arg Thr Ile Ser Phe Asp Ile Pro Pro Gln Glu Ile Leu Thr Lys Asp100 105 110 Ser Val Thr Ile Ser Val Asp Gly Val Val Tyr Tyr Arg Val GlnAsn 115 120 125 Ala Thr Leu Ala Val Ala Asn Ile Thr Asn Ala Asp Ser AlaThr Arg 130 135 140 Leu Leu Ala Gln Thr Thr Leu Arg Asn Val Leu Gly ThrLys Asn Leu 145 150 155 160 Ser Gln Ile Leu Ser Asp Arg Glu Glu Ile AlaHis Asn Met Gln Ser 165 170 175 Thr Leu Asp Asp Ala Thr Asp Ala Trp GlyIle Lys Val Glu Arg Val 180 185 190 Glu Ile Lys Asp Val Lys Leu Pro ValGln Leu Gln Arg Ala Met Ala 195 200 205 Ala Glu Ala Glu Ala Ser Arg GluAla Arg Ala Lys Val Ile Ala Ala 210 215 220 Glu Gly Glu Met Asn Ala SerArg Ala Leu Lys Glu Ala Ser Met Val 225 230 235 240 Ile Thr Glu Ser ProAla Ala Leu Gln Leu Arg Tyr Leu Gln Thr Leu 245 250 255 Thr Thr Ile AlaAla Glu Lys Asn Ser Thr Ile Val Phe Pro Leu Pro 260 265 270 Ile Asp MetLeu Gln Gly Ile Ile Gly Ala Lys His Ser His Leu Gly 275 280 285 280amino acids amino acid single linear GenBank 1065452 4 Met Asn Leu LysThr Cys Ser Leu Ser Thr His Ser Phe Leu Gln Lys 1 5 10 15 Lys Asn GluLys His Asp Gly Asn Pro Glu His Tyr Asp Thr Gly Leu 20 25 30 Gly Phe CysGly Trp Phe Leu Met Gly Leu Ser Trp Ile Met Val Ile 35 40 45 Ser Thr PhePro Val Ser Ile Tyr Phe Cys Met Lys Val Val Gln Glu 50 55 60 Tyr Glu ArgAla Val Ile Phe Arg Leu Gly Arg Leu Ile Gly Gly Gly 65 70 75 80 Ala LysGly Pro Gly Ile Phe Phe Val Leu Pro Cys Ile Glu Ser Tyr 85 90 95 Thr LysVal Asp Leu Arg Thr Val Ser Phe Ser Val Pro Pro Gln Glu 100 105 110 IleLeu Thr Lys Asp Ser Val Thr Thr Ser Val Asp Ala Val Ile Tyr 115 120 125Tyr Arg Ile Ser Asn Ala Thr Val Ser Val Ala Asn Val Glu Asn Ala 130 135140 His His Ser Thr Arg Leu Leu Ala Gln Thr Thr Leu Arg Asn Met Leu 145150 155 160 Gly Thr Arg Ser Leu Ser Glu Ile Leu Ser Asp Arg Glu Thr LeuAla 165 170 175 Ala Ser Met Gln Thr Ile Leu Asp Glu Ala Thr Glu Ser TrpGly Ile 180 185 190 Lys Val Glu Arg Val Glu Ile Lys Asp Val Arg Leu ProIle Gln Leu 195 200 205 Gln Arg Ala Met Ala Ala Glu Ala Glu Ala Thr ArgGlu Ala Arg Ala 210 215 220 Lys Val Ile Ala Ala Glu Gly Glu Gln Lys AlaSer Arg Ala Leu Arg 225 230 235 240 Asp Ala Ala Ser Val Ile Ala Gln SerPro Ala Ala Leu Gln Leu Arg 245 250 255 Tyr Leu Gln Thr Leu Asn Ser ValAla Arg Glu Lys Phe Asp Asp His 260 265 270 Leu Pro Thr Ser Asp Gly IleSer 275 280 415 amino acids amino acid single linear GenBank 1353669 5Met Glu Tyr Gly Met Pro Glu Gly Ser Tyr Asp Ser Val Phe Thr Tyr 1 5 1015 Ala Pro Tyr Asn Asp Leu Asp Lys Met Gly Tyr Met Gly Pro Ala Arg 20 2530 Gln Gly Met Met Leu Gly Asn Lys Tyr Gly Asn Phe Thr Tyr Thr Arg 35 4045 Asp Tyr Gly Val Asn Met Glu Asp Asp Ile Lys Pro Leu Ser Ala Ile 50 5560 Glu Leu Leu Ile Phe Cys Val Ser Phe Leu Phe Val Val Met Thr Met 65 7075 80 Pro Leu Ser Leu Leu Phe Ala Leu Lys Phe Ile Ser Thr Ser Glu Lys 8590 95 Leu Val Val Leu Arg Leu Gly Arg Ala Gln Lys Thr Arg Gly Pro Gly100 105 110 Ile Thr Leu Val Ile Pro Cys Ile Asp Thr Thr His Lys Val ThrMet 115 120 125 Ser Ile Thr Ala Phe Asn Val Pro Pro Leu Gln Ile Ile ThrThr Asp 130 135 140 Arg Gly Leu Val Glu Leu Gly Ala Thr Val Phe Leu LysIle Arg Asp 145 150 155 160 Pro Ile Ala Ala Val Cys Gly Val Gln Asp ArgAsn Ala Ser Val Arg 165 170 175 Thr Leu Ala Asn Thr Met Leu Tyr Arg TyrIle Ser Lys Lys Arg Ile 180 185 190 Cys Asp Val Thr Ser Ser Gln Asp ArgArg Ile Ile Ser Ala Asn Leu 195 200 205 Lys Asp Glu Leu Gly Ser Phe ThrCys Gln Phe Gly Val Glu Ile Thr 210 215 220 Asp Val Glu Ile Ser Asp ValLys Ile Val Lys Glu Gly Glu Asn Met 225 230 235 240 Gly Met Ser Ala LeuSer Ser Val Ala Lys Ser Asp Ala Gly Gln Gln 245 250 255 Leu Trp Gln ValIle Gly Pro Val Phe Glu Asp Phe Ala Lys Glu Cys 260 265 270 Ala Ala GluGlu Lys Ala Lys Glu Asn Ala Pro Leu Val Asp Leu Ser 275 280 285 Asp ValPro Ser Thr Ser Ala Ala Gly Thr Ser Thr Asp Thr Pro Asn 290 295 300 IlePro Ser Ile Asp Ile Asp His Leu Ile Ser Val Ala Ser Leu Ala 305 310 315320 Met Asp Glu His Leu Val Arg Leu Ile Gly Arg Val Phe Gln Ile Asn 325330 335 Cys Lys Asp Ile Glu Pro Ile Cys Ile Asp Leu Lys His Gly Ser Gly340 345 350 Ser Ala Tyr Lys Gly Thr Ser Leu Asn Pro Asp Val Val Phe GluThr 355 360 365 Ser Leu Glu Val Phe Gly Lys Ile Leu Thr Lys Glu Val SerPro Val 370 375 380 Thr Val Tyr Met Asn Gly Asn Leu Lys Val Lys Gly SerIle Gln Asp 385 390 395 400 Ala Met Gln Leu Lys His Leu Val Glu Arg MetSer Asp Trp Leu 405 410 415 381 amino acids amino acid single linear Owl79701 6 Met Gln Gly Ala Val Ala Gly Leu Val Phe Leu Ala Val Leu Val Ile1 5 10 15 Phe Ala Ile Ile Val Val Ala Lys Ser Val Ala Leu Ile Pro GlnAla 20 25 30 Glu Ala Ala Val Ile Glu Arg Leu Gly Arg Tyr Ser Arg Thr ValSer 35 40 45 Gly Gln Leu Thr Leu Leu Val Pro Phe Ile Asp Arg Val Arg AlaArg 50 55 60 Val Asp Leu Arg Glu Arg Val Val Ser Phe Pro Pro Gln Pro ValIle 65 70 75 80 Thr Glu Asp Asn Leu Thr Leu Asn Ile Asp Thr Val Val TyrPhe Gln 85 90 95 Val Thr Val Pro Gln Ala Ala Val Tyr Glu Ile Ser Asn TyrIle Val 100 105 110 Gly Val Glu Gln Leu Thr Thr Thr Thr Leu Arg Asn ValVal Gly Gly 115 120 125 Met Thr Leu Glu Gln Thr Leu Thr Ser Arg Asp GlnIle Asn Ala Gln 130 135 140 Leu Arg Gly Val Leu Asp Glu Ala Thr Gly ArgTrp Gly Leu Arg Val 145 150 155 160 Ala Arg Val Glu Leu Arg Ser Ile AspPro Pro Pro Ser Ile Gln Ala 165 170 175 Ser Met Glu Lys Gln Met Lys AlaAsp Arg Glu Lys Arg Ala Met Ile 180 185 190 Leu Thr Ala Glu Gly Thr ArgGlu Ala Ala Ile Lys Gln Ala Glu Gly 195 200 205 Gln Lys Gln Ala Gln IleLeu Ala Ala Glu Gly Ala Lys Gln Ala Ala 210 215 220 Ile Leu Ala Ala GluAla Asp Arg Gln Ser Arg Met Leu Arg Ala Gln 225 230 235 240 Gly Glu ArgAla Ala Ala Tyr Leu Gln Ala Gln Gly Gln Ala Lys Ala 245 250 255 Ile GluLys Thr Phe Ala Ala Ile Lys Ala Gly Arg Pro Thr Pro Glu 260 265 270 MetLeu Ala Tyr Gln Tyr Leu Gln Thr Leu Pro Glu Met Ala Arg Gly 275 280 285Asp Ala Asn Lys Val Trp Val Val Pro Ser Asp Phe Asn Ala Ala Leu 290 295300 Gln Gly Phe Thr Arg Leu Leu Gly Lys Pro Gly Glu Asp Gly Val Phe 305310 315 320 Arg Phe Glu Pro Ser Pro Val Glu Asp Gln Pro Lys His Ala AlaAsp 325 330 335 Gly Asp Asp Ala Glu Val Ala Gly Trp Phe Ser Thr Asp ThrAsp Pro 340 345 350 Ser Ile Ala Arg Ala Val Ala Thr Ala Glu Ala Ile AlaArg Lys Pro 355 360 365 Val Glu Gly Ser Leu Gly Thr Pro Pro Arg Leu ThrGln 370 375 380 199 amino acids amino acid single linear GenBank 15915147 Met Lys Val Asn Asp Met Phe Trp Phe Trp Leu Ile Leu Gly Ile Ile 1 5 1015 Ala Leu Phe Ile Ile Val Lys Ala Ile Val Ile Val Asn Gln Tyr Glu 20 2530 Gly Gly Leu Ile Phe Arg Leu Gly Arg Val Ile Gly Lys Leu Lys Pro 35 4045 Gly Ile Asn Ile Ile Ile Pro Phe Leu Asp Val Pro Val Lys Val Asp 50 5560 Met Arg Thr Arg Val Thr Asp Ile Pro Pro Gln Glu Met Ile Thr Lys 65 7075 80 Asp Asn Ala Val Val Lys Val Asp Ala Val Val Tyr Tyr Arg Val Ile 8590 95 Asp Val Glu Lys Ala Ile Leu Glu Val Glu Asp Tyr Glu Tyr Ala Ile100 105 110 Ile Asn Leu Ala Gln Thr Thr Leu Arg Ala Ile Ile Gly Ser MetGlu 115 120 125 Leu Asp Glu Val Leu Asn Lys Arg Glu Tyr Ile Asn Ser LysLeu Leu 130 135 140 Glu Ile Leu Asp Arg Glu Thr Asp Ala Trp Gly Val ArgIle Glu Lys 145 150 155 160 Val Glu Val Lys Glu Ile Asp Pro Pro Glu AspIle Lys Asn Ala Met 165 170 175 Ala Gln Gln Met Lys Ala Glu Arg Leu LysArg Ala Ala Ile Leu Glu 180 185 190 Ala Glu Gly Glu Lys Pro Glu 195

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence of SEQ ID No: 1, b) a naturallyoccurring polypeptide comprising an amino acid sequence at least 90%identical to an amino acid sequence of SEQ ID No: 1, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence of SEQ IDNo: 1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence of SEQ ID No:
 1. 2. An isolated polypeptide of claim 1,having a sequence of SEQ ID No:
 1. 3. An isolated polynucleotideencoding a polypeptide of claim
 1. 4. An isolated polynucleotideencoding a polypeptide of claim
 2. 5. An isolated polynucleotide ofclaim 4, having a sequence of SEQ ID No:
 2. 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. A transgenic organism comprising arecombinant polynucleotide of claim
 6. 9. A method for producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide has the sequence of SEQ ID No:
 1. 11. An isolated antibodywhich specifically binds to a polypeptide of claim
 1. 12. An isolatedpolynucleotide comprising a sequence selected from the group consistingof: a) a polynucleotide comprising a polynucleotide sequence of SEQ IDNo: 2, b) a naturally occurring polynucleotide comprising apolynucleotide sequence at least 90% identical to a polynucleotidesequence of SEQ ID No: 2, c) a polynucleotide having a sequencecomplementary to a polynucleotide of a), d) a polynucleotide having asequence complementary to a polynucleotide of b) and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID No:
 1. 19. A method for treating a disease orcondition associated with decreased expression of functional integralmembrane protein, comprising administering to a patient in need of suchtreatment the composition of claim
 17. 20. A method for screening acompound for effectiveness as an agonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting agonist activity in the sample.21. A composition comprising an agonist compound identified by a methodof claim 20 and a pharmaceutically acceptable excipient.
 22. A methodfor treating a disease or condition associated with decreased expressionof functional integral membrane protein, comprising administering to apatient in need of such treatment a composition of claim
 21. 23. Amethod for screening a compound for effectiveness as an antagonist of apolypeptide of claim 1, the method comprising: a) exposing a samplecomprising a polypeptide of claim 1 to a compound, and b) detectingantagonist activity in the sample.
 24. A composition comprising anantagonist compound identified by a method of claim 23 and apharmaceutically acceptable excipient.
 25. A method for treating adisease or condition associated with overexpression of functionalintegral membrane protein, comprising administering to a patient in needof such treatment a composition of claim
 24. 26. A method of screeningfor a compound that specifically binds to the polypeptide of claim 1,said method comprising the steps of: a) combining the polypeptide ofclaim 1 with at least one test compound under suitable conditions, andb) detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 12, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, said method comprising: a) treating a biologicalsample containing nucleic acids with the test compound; b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of integral membrane protein in a biological samplecomprising the steps of: a) combining the biological sample with anantibody of claim 11, under conditions suitable for the antibody to bindthe polypeptide and form an antibody:polypeptide complex; and b)detecting the complex, wherein the presence of the complex correlateswith the presence of the polypeptide in the biological sample.
 31. Theantibody of claim 11, wherein the antibody is: a) a chimeric antibody,b) a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, ore) a humanized antibody.
 32. A composition comprising an antibody ofclaim 11 and an acceptable excipient.
 33. A method of diagnosing acondition or disease associated with the expression of integral membraneprotein in a subject, comprising administering to said subject aneffective amount of the composition of claim
 32. 34. A composition ofclaim 32, wherein the antibody is labeled.
 35. A method of diagnosing acondition or disease associated with the expression of integral membraneprotein in a subject, comprising administering to said subject aneffective amount of the composition of claim
 34. 36. A method ofpreparing a polyclonal antibody with the specificity of the antibody ofclaim 11 comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence of SEQ ID No: 1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response; b) isolatingantibodies from said animal; and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having an amino acid sequence of SEQID No:
 1. 37. An antibody produced by a method of claim
 36. 38. Acomposition comprising the antibody of claim 37 and a suitable carrier.39. A method of making a monoclonal antibody with the specificity of theantibody of claim 11 comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID No: 1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID No:
 1. 40. A monoclonal antibody produced by a method of claim39.
 41. A composition comprising the antibody of claim 40 and a suitablecarrier.
 42. The antibody of claim 11, wherein the antibody is producedby screening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method for detecting a polypeptide havingan amino acid sequence of SEQ ID No: 1 in a sample, comprising the stepsof: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID No: 1 in the sample.
 45. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID No: 1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide; and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID No: 1.